The applicant, Dr. Sarah Parker, will conduct research on phospholipases in the pathogenesis of Mycobacterium tuberculosis. Though M. tuberculosis kills three million people per year, 450,000 of whom are children, the mechanisms of its success are not well understood. Evidence suggests that phospholipase A2 (PLA2), which is described here for the first time in M. tuberculosis, and phospholipase C (PLC) are surface exposed components of the mycobacterial cell wall. These phospholipases degrade phospholipids to fatty acids and other inflammatory mediators, and Dr. Parker hypothesizes that they contribute to the intracellularsurvival or extracellular pathogenesis of M. tuberculosis. She postulates that the degradation of hostsurfactant and membranes by M. tuberculosis PLC and PLA2 contributes to virulence by a number of possible mechanisms: facilitation of bacillary entry into host cells, production of inflammatory mediators, alteration of the phagosomal environment, and/or acquisition of nutrition in the form of fatty acids. Dr. Parker has designed her specific aims to address each of these possible contributions of PLAand PLC to M. tuberculosis pathogenesis. Currently she has a partial PLC deletion mutant in M. tuberculosis. Once she has purified PLA2 from M. tuberculosis, she will use the partial amino acid sequence to identify the pla2 gene. She will construct phospholipase C, A2 and C + A2 deletion mutants, and they will be bacteriologically and biochemically characterized. Phospholipid degradation and utilization by these strains will be investigated in varied nutritional environments, including using pulmonary surfactant as a carbon source. In a human macrophage model of infection, she will analyze PLC and PLA2 contributions to bacillary macrophage entry, trafficking and survival. The research project is designed to result in Dr. Parker becoming a successful and independent M. tuberculosis investigator. Not only will she learn many basic and state-of-the-art techniques, she will receive didactic training to compliment her research. She will spend 15% of her time honing her clinical skills as an assistant professor in Pediatric Infectious Diseases. Dr. Parker is committed to research in M. tuberculosis, and it holds a special significance to her because of her life experiences.